Antimicrobial and quorum sensing inhibitory activity of epiphytic bacteria isolated from the red alga Halymenia durvillei

Halymenia durvillei is a red alga that is commonly utilized in the Philippines as food and as a source of high-value natural products for industrial applications. However, there are no studies regarding the microbial community associated with H. durvillei and its potential applications. This study aimed to isolate and identify the epiphytic bacteria of H. durvillei and determine their antimicrobial and quorum sensing inhibitory (QSI) effects. The thalli of H. durvillei were collected at the shores of Santa Fe, Bantayan, Cebu, Philippines. Bacterial isolates were identified using 16S rRNA, and their ethyl acetate (EtOAc) extracts were subjected to antimicrobial susceptibility tests against representative species of yeast and Gram-negative and Gram-positive bacteria. Their QSI activity against Chromobacterium violaceum was also determined. Fourteen distinct bacterial colonies belonging to four genera, namely Alteromonas (3), Bacillus (5), Oceanobacillus (1) and Vibrio (5), were successfully isolated and identified. All 14 bacterial isolates exhibited antibacterial effects. EPB9, identified as Bacillus safensis , consistently showed the strongest inhibition against Escherichia coli , Staphylococcus aureus and Staphylococcus epidermidis , with minimum inhibitory concentrations (MICs) ranging from 0.0625 to 1.0 mg ml−1. In contrast, all 14 isolates showed weak antifungal effects. Both B. safensis (EPB9) and Bacillus australimaris (EPB15) exhibited QSI effects at 100 mg ml−1, showing opaque zones of 3.1±0.9 and 3.8±0.4 mm, respectively. This study is the first to isolate and identify the distinct microbial epiphytic bacterial community of H. durvillei and its potential as an abundant resource for new antibacterial and QSI bioactives.


INTRODUCTION
The search for novel bioactive compounds in developing new drugs has prompted the exploration of underutilized resources in various ecosystems and environmental conditions [1].Marine microbes, for example, have gained the attention of many researchers due to the chemical and structural diversity of the metabolites they produce and the various modes of action they exhibit [2].Drug candidates that were purified and characterized from marine microbial symbionts showed potent bioactivities against various pathogens and diseases, including antibiotic-resistant micro-organisms, diabetes, cancer and inflammation [2][3][4][5].However, the potential use of marine resources in developing new pharmaceuticals is still underexplored and presents a new area for natural product research.
The emergence of antimicrobial-resistant (AMR) pathogens is one of the most pressing public health concerns worldwide.Several antibiotics are no longer effective in treating these resistant strains, posing a substantial threat to millions of lives.Moreover, temperature changes due to global warming could lead to rapid increases in AMR bacteria because of increased bacterial growth rates and horizontal gene transfer [6].Increasing temperatures lead to increased human and animal contact, facilitating the spread of pathogens that harbour new resistance mechanisms [7].Despite the imminent threat that antimicrobial resistance poses, efforts in antibiotic research have declined in the past decades [8].Thus, finding new antibiotics and developing new strategies for managing antimicrobial resistance is imperative.
Quorum sensing (QS) is a type of bacterial cell-cell communication that is commonly associated with the release of signals that regulate virulence and pathogenicity-related genes.Inhibition of QS has been shown to reduce the pressure to produce drug resistance in bacteria by limiting virulence and pathogenicity [9].Chromobacterium violaceum is a Gram-negative bacillus that is commonly found in soil and freshwater.It is generally regarded as an opportunistic pathogen commonly affecting immunocompromised individuals [10,11].The production of violacein, a purple pigment produced by C. violaceum, is regulated through QS.Thus, the production of violacein is a valuable predictor of quorum sensing inhibitory (QSI) activity, as it is easy to visualize and quantify [12].In addition, the production of cyanide and elastase in C. violaceum is heavily controlled through QS [10].Taken together, these characteristics of C. violaceum allow it to be a suitable reporter strain for QSI screening.
Resazurin is a dye that changes colour from blue to pink when reduced to resorufin due to aerobic respiration of the active microbial cells.Thus, colorimetric assays utilizing the oxidation-reduction of resazurin have been widely employed in the determination of the least inhibitory concentration that could inhibit bacterial growth.This method has been found to be rapid, objective, scalable, quantitative and cost-effective, and, more importantly, it has been endorsed by the World Health Organization for the colorimetric liquid culture-based drug susceptibility assay for Mycobacterium tuberculosis [13][14][15][16].The use of resazurin for minimum inhibitory concentration (MIC) determination has been successfully validated for its accuracy using the performance of standard antibiotics against several American Type Culture Collection (ATCC) strains and, in comparison, with those published by the Clinical and Laboratory Standards Institute (CSLI) [13][14][15]17].
Microbial communities on the surface of macroalgae exhibit a symbiotic interaction that is crucial for the development and defence of their host alga [18].Selvarajan et al. [19] reported that host-specific microbial communities are present in seaweeds, which are greatly influenced by the surface chemistry of the algal host.These epiphytic communities confer protection by producing antifouling and antimicrobials against secondary colonization of pathogenic microscopic and macroscopic epibiota, since macroalgae have no immune system [20].This implies that marine microbes are an abundant resource of natural products that can be explored for their bioactivities.
Halymenia durvillei is a red alga ubiquitously distributed in the Indo-Pacific region.It is commonly known as 'red sea lettuce' and is characterized as having a dark red colour, branching blades with toothed margins and a firm gelatinous texture [21,22].In the Philippines, there are ongoing efforts to cultivate H. durvillei as a potential source of high-value pigments, phycoerythrin and r-phycocyanin [19].Polysaccharides from H. durvillei, such as sulfated galactans, can be used in different applications in food technology, cosmetics and pharmaceutical research [23,24].Although the potential industrial applications of H. durvillei are well established, there are no studies regarding the distinct bacterial community of the algae.
This study aimed to isolate and identify the epiphytic bacterial community in the red alga, H. durvillei, and screen for their antimicrobial and QSI activities against different human pathogenic micro-organisms.

Collection and identification of H. durvillei
The thalli of H. durvillei were collected from the intertidal area at Santa Fe, Bantayan Island, Cebu, Philippines (11°09′21″N, 123°48′30″W).The collected samples were kept cool during transport and were immediately processed upon arrival at the University of San Carlos (USC) Applied Microbiology Laboratory.Morphological and molecular identification of the collected specimens was performed, and the voucher specimens were deposited at the USC Herbarium.

Isolation of epiphytic bacteria from H. durvillei
The thalli of H. durvillei were washed with sterile filtered seawater and rinsed three times with sterile phosphate-buffered saline (PBS) to remove debris and loosely attached bacteria from the samples.The thalli were then swabbed with a sterile cotton applicator and inoculated in Zobell marine agar supplemented with nystatin (20 µg ml −1 ) (HiMedia, Mumbai, India).The cultures

Impact Statement
The bacterial symbionts of many marine eukaryotes are known to produce chemically diverse metabolites with various bioactivities.This paper shows that the epiphytic bacterial community associated with H. durvillei is rich in antimicrobial metabolites and can potentially be used as a natural therapy against clinically significant pathogens.
were incubated at 25 °C for 5 days.The colonies showing distinct morphologies were picked and streaked to isolate individual colonies [25].

Colony morphology and biochemical characterization of bacterial isolates
The colony morphology of the bacterial isolates was noted based on form, elevation and margin.Phenotypic characterization of all 14 isolates was performed with identification as described in Bergey's Manual of Systematic Bacteriology [26,27].Colony morphology for purified microbial isolates was determined using a Zeiss stereomicroscope (Carl Zeiss NTS Ltd, Germany), and the biochemical reactions were screened using analytical profile index (API) 20E strips (bioMérieux, France) for the characterization of isolates belonging to the Firmicutes group [28,29].Biochemical tests were conducted in accordance with the standard operating procedure per the manufacturer's instructions.Briefly, bacterial isolates were grown on plates of marine agar overnight.A bacterial suspension was made using 5 ml of sterile saline solution after 24 h, and it was adjusted until it was comparable to the 0.5 McFarland standard with roughly 1.5×10 8 c.f.u.ml −1 microbial cells.Then, 200 µl of bacterial suspension was added to each of the API strip compartments.After 24 h of incubation at 37 °C, the results were read and interpreted using the reading table provided in the manufacturer's manual.A drop of 10 % ferric chloride (FeCl 3 ) was added for the TDA test, Kovacs reagent was added for the IND test, and 40 % potassium hydroxide (KOH) (VP1) and α -naphthol (VP 2) were added for the VP test.All reactions were recorded as (+) for positive reactions and (−) for negative reactions.

Molecular identification of bacterial isolates
Pure colonies were grown in nutrient media and harvested for genomic DNA extraction using the NucleoSpin Microbial DNA Mini kit (Macherey-Nagel, PA, USA).The 16S rRNA of each bacterial isolate was amplified by polymerase chain reaction (PCR) using the primers 27F(5' AGAGTTTGATCCGGCTCAG-3′) and 1492R(5′-GGTTACCTTGTTACGACTT-3′) [30].The final volume of the PCR mix for each reaction was 25 µl containing TaKaRa Ex Taq DNA polymerase, Ex Taq reaction buffer (Mg2+free), magnesium chloride (MgCl2), sterile distilled water, 10 pmol of each primer and 1-10 ng of template DNA.The PCR conditions were as follows: initial denaturation at 94 °C for 2 min, 35 cycles at 94 °C for 1 min, annealing at 55-72 °C for 1.5 min, elongation at 72 °C for 1 min and final extension at 72 °C for 3 min [31].The PCR products were electrophoresed in 1 % agarose gel and visualized under a UV transilluminator.The PCR products were sent to Macrogen (Republic of Korea) for gel extraction and DNA sequencing.The Sequencher 5.4.6 program was used to analyse the sequences gathered.The isolated bacteria were identified by comparing the consensus sequences to reference bacterial sequences using the blastn program (http://www.ncbi.nlm.nih.gov).

Field emission scanning electron microscopy (FESEM)
The bacterial isolates that exhibited consistent antibiotic and QSI activities were further imaged using FESEM [32].Bacillus safensis EPB9 and Bacillus australimaris EPB15 were grown in a 24-well microplate for 24 h using Zobell marine broth as the medium.The cells were allowed to attach to sterile glass coverslips submerged in the wells.Afterward, the coverslips were removed and washed twice with PBS.The samples were fixed in 2.5 % glutaraldehyde, followed by dehydration using different concentrations of ethanol (10-100 %) and critical point drying (Samdri PVT-3D, Tousimis, USA).The samples were stored in a desiccator and then coated twice with gold using a Quorum Q150R ion sputter coater (Quorum Technologies Ltd, UK).The surfaces of the bacterial samples were visualized using a Gemini Sigma 500 VP FESEM (Zeiss, USA) in high-vacuum mode at 3.0-3.5 kV.

Preparation of crude extracts from bacterial isolates
The broth cultures of each bacterial isolate grown in 250 ml Zobell marine broth (25 °C, 7 days) were extracted with ethyl acetate (EtOAc).The organic layer was collected and concentrated to dryness using a rotary evaporator at 35 °C.

Antimicrobial screening
The EtOAc extracts of the bacterial isolates were screened for their antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus BIOTECH 1664 and Staphylococcus epidermidis BIOTECH 1800), Gram-negative bacteria (Escherichia coli BIOTECH 1634 and Serratia marcescens BIOTECH 1784) and yeast (Candida albicans BIOTECH 2085).All micro-organisms were acquired from the BIOTECH Philippine National Collection of Microorganisms and are not classified as MDR bacteria.Bacterial and fungal test micro-organisms were grown on Mueller-Hinton agar (HiMedia, Mumbai, India) and potato dextrose agar (HiMedia, Mumbai, India), respectively.The EtOAc extracts were dissolved in dimethyl sulfoxide (DMSO) at 100 mg ml −1 .The antimicrobial activity of the extracts was determined using the disc diffusion method in accordance with the Clinical and Laboratory Standards Institute (CSLI) recommendation [33].Briefly, the inoculum containing a 24 h culture of each test microorganism, adjusted until comparable to 0.5 McFarland standard, was swabbed on agar plates.The 6 mm discs containing the test extracts were placed on top of the media and incubated for 24 h at 25 °C.The zones of inhibition for each disc were measured after that.The positive controls used for antibacterial and antifungal screening were streptomycin and fungisol, respectively.The CSLI classified the test pathogens (S. aureus, S. epidermidis, E. coli and S. marcescens) as being susceptible to the antibiotic standard streptomycin [33].

Determination of MIC and minimum bactericidal concentration (MBC)
The MIC and MBC of the extracts were determined using the broth microdilution method with some modifications [17].Briefly, inocula of 24 h cultures of test micro-organisms were adjusted to an absorbance value of 0.063, which is equivalent to an absorbance value of 0.5 McFarland standard (10 8 c.f.u ml −1 ) [34], and then serially diluted to 10 5 c.f.u ml −1 (OD 600 ) and inoculated in a microplate.The extracts were added in concentrations of 0.0625-8 mg ml −1 , and the cultures were incubated for 24 h at 25 °C.Afterward, 10 µl of 0.015 % resazurin was added to each well, followed by an incubation period of 4 h.The resazurin dye indicates the presence of active bacterial cells and its colour changes upon a redox reaction caused by active cell metabolism.A colour change from blue to pink after adding the dye signifies the presence of active bacteria.The MIC value per extract was assigned to the lowest concentration, where a colour change was not observed (blue).The MBC value was obtained by plating the contents of the well with the highest and lowest MIC values (blue wells) into sterile Mueller-Hinton agar.Concentrations where bacteria did not grow served as the MBC.Streptomycin was used as a positive control at 10 µg ml −1 .

QSI activity
The agar disc diffusion method was performed using the CLSI protocol with slight modifications to screen for the QSI activity of the crude extracts [33,34].C. violaceum NCTC 9757 was cultured in Luria-Bertani (LB) agar [1 g tryptone, 0.5 g yeast extract, 1 g sodium chloride (NaCl), 2 g peptone and 1.5 g in 100 distilled water] and incubated for 24 h at 28 °C.Standardized bacterial suspension with 1.5×10 8 c.f.u ml −1 (OD 600 ) was prepared.The inoculum was uniformly swabbed in LB agar plates.N-acyl homoserine lactones inducer (80 mM) was then swabbed as a second layer.Fifteen microlitres of the 100 mg ml −1 crude extract were loaded into sterile discs (6 mm diameter; Whatman no. 1).The prepared discs were placed on the swabbed plates aseptically.The inoculated plates were then incubated for 24 h at 28 °C.The diameters of the opaque and clear zones were then measured using a microcaliper.QSI was then calculated using the following formula: wherein r2 is the total growth and the QS inhibition zone radius and r1 is the clear zone radius.Cinnamaldehyde (10 mg ml −1 ) and DMSO were used as the positive and negative controls, respectively.

Statistical analysis
IBM SPSS statistics software (trial version) (Illinois, USA) was used for statistical analysis.The nonparametric Kruskal-Wallis test was performed, followed by a pairwise comparison test adjusted by the Bonferroni correction for multiple tests.

Isolation, biochemical characterization and molecular identification of H. durvillei-associated epiphytic bacteria
The epiphytic bacteria associated with H. durvillei were isolated by swabbing the surface of its thallus and inoculating it on marine agar.Colonies that showed distinct morphologies based on form, elevation and margin were picked and purified further.Of note, several of the bacterial colonies exhibited clear zones around them, indicating some degree of antimicrobial activity (Fig. 1a).The 14 bacterial colonies that were successfully isolated were identified by comparing their consensus 16S rRNA gene sequences with reference sequences on blastn [National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov].Fourteen colonies were successfully identified with percentage identities >99.51 %.The identified bacteria belong to the genera Alteromonas, Bacillus, Oceanobacillus and Vibrio (Table 1).The colony morphology of 14 bacterial isolates is summarized in Table 2.The bacterial structure and morphology of two bacterial isolates showing both antibacterial and QSI activity, B. safensis (EPB9) and B. australimaris (EPB15), were viewed under FESEM.B. safensis (EPB9) was shown as a single cell (Fig. 1c), whereas B. australimaris (EPB15) was seen as an aggregate of cells embedded in an extracellular matrix (Fig. 1d).In addition, biochemical characterization of bacterial isolates belonging to the Firmicutes group was performed to further determine their identity (Table 3).In particular, the results showed that both B. safensis (EPB9) and B. australimaris (EPB15) were Gram-positive and rod-shaped cells with colonies in circular form with curled margins and wrinkled elevations.This colonial morphology is in contrasts to that found by Singh et al. [35], who had an entire margin and raised elevation for their B. safensis AS08 isolate.However, a similar biochemical profile was observed, as it was positive for catalase, oxidase, ß-galactosidase and gelatin hydrolysis.Based on morphological and biochemical characteristics, the bacterial isolates EPB9 and EPB15 were determined to be Bacillus species.The 16S rRNA gene sequencing further confirmed the phenotypic characterization of these two isolates as being from the phylum Firmicutes.

Antimicrobial screening
The EtOAc extracts of the identified epiphytic bacteria were screened for their antimicrobial activities against representative species of yeast (C.albicans), Gram-negative (E. coli and S. marcescens) and Gram-positive (S. aureus and S. epidermidis) bacteria using the agar disc diffusion method for antibiotic susceptibility testing (Table 4).The extracts of all 14 bacteria showed antibiotic effects at 100 mg ml −1 against Gram-negative and Gram-positive test bacteria.EPB9, identified as B. safensis, showed consistently strong antibiotic effects against the test bacteria (zone of inhibition >10 mm), wherein higher zones of inhibition compared with the positive control, streptomycin, were observed in E. coli and S. epidermidis (Fig. 2b).The extracts were analysed further to  determine their MICs and MBCs against the test bacteria.In contrast, only three isolates (EPB11, EPB12 and EPB17) showed inhibition against C. albicans, with all three exhibiting weak antifungal effects compared to the positive control.

MIC and MBC of the isolated bacteria from H. durvillei
The MICs and MBCs of the EtOAc extracts of the bacterial isolates that showed consistently strong antimicrobial activities against the test bacteria were determined (Table 5).The colorimetric reaction observed in each well after the addition of resazurin was used to determine the MIC and MBC of each extract (Fig. 2c).EPB9 showed a very low MIC for E. coli (0.0625 mg ml −1 ), S. aureus (1 mg ml −1 ) and S. epidermidis (1 mg ml −1 ).The rest of the extracts exhibited MICs against the test bacteria ranging from 0.25-8 mg ml −1 .

QSI activity
The QSI effects of the EtOAc extracts of the bacterial isolates against C. violaceum were determined using the agar disc diffusion method (Fig. 2a).B. safensis EPB9 and B. australimaris EPB15 exhibited QSI effects at 100 mg ml −1 .Both extracts showed higher QSI activity than the positive control, cinnamaldehyde (Table 6).The rest of the extracts did not exhibit any QSI activity.

DISCUSSION
The host seaweed and its microbiota are in such a close association that they act like a single unified organism, otherwise known as a holobiont [18].Evidence suggests that seaweed microbiota is host-specific and greatly influenced by algal metabolites and surface architecture [36].Several studies examining the microbial epibiota of different marine seaweeds have shown that the major phyla on their surfaces are Proteobacteria, Firmicutes and Bacteriodetes [18,37].Moreover, these microbes are predominantly Gram-negative and exhibit motility, indicating that they form biofilms.Motile bacteria respond to stressful situations using negative chemotaxis (e.g.swarming away), resulting in the transition from the unicellular and planktonic stage to the biofilm stage of bacterial growth and loss of flagellar motility.The activation of negative chemotaxis acts as a catalyst for the development of biofilms and other defence mechanisms against unfavourable environmental conditions [38,39].The signalling molecule c-di-GMP in Gram-negative bacteria inhibits flagellar motility and encourages the production of adhesion factors and extracellular polymeric substances (EPSs) for biofilm formation [40].A study reported that the 31 phylotypes of epiphytic bacteria associated with H. floresii are primarily composed of groups from Proteobacteria (84 %), Bacteriodetes (13 %) and Firmicutes (3 %) [41].
Consistent with these studies, our results show that most of the bacterial isolates from H. durvillei are Gram-negative and are members of the group Proteobacteria (57 %), except for six species belonging to Firmicutes (43 %) (Table 1).The members of the group Bacteriodetes were not identified in this study.In addition, the bacterial isolates from H. durvillei belong to four genera: Alteromonas, Bacillus, Oceanobacillus and Vibrio.Among them, members of the genera Vibrio and Alteromonas are reported to cause diseases in marine eukaryotes such as fishes, crustaceans, corals and seaweeds [42,43].Interestingly, these species of bacteria were isolated from the healthy thalli of H. durvillei.Studies regarding the interactions between these bacterial isolates and their host alga can be further explored to elucidate their pathogenicity.In contrast, there are no reports regarding the pathogenicity of Bacillus altitudinis, B. australimaris, B. safensis and Oceanobacillus to marine organisms.Furthermore, this study only identified bacterial species that could grow in laboratory conditions.It did not consider all the micro-organisms present in H. durvillei.To achieve a more comprehensive examination of the microbial flora of H. durvillei, a metagenomics approach is needed.
The epibiota of marine seaweeds are reported to deter the attachment of other surface colonizers by producing antimicrobials and antifouling metabolites [36].Several studies have shown that extracts from the epiphytic bacteria of various seaweed species exhibit potent in vitro antimicrobial activity against a wide range of human pathogenic bacteria [44,45].All 14 bacterial isolates from H. durvillei exhibited antibiotic effects against representative Gram-negative and Gram-positive bacteria species.In particular, V. owensii (EPB1), V. owensii (EPB3), V. rotiferianus (EPB4), Alteromonas sp.(EPB7), Alteromonas sp.(EPB17) and B. safensis (EPB9) displayed the strongest and most consistent activity among the bacterial isolates.V. owensii and V. rotiferianus are welldocumented pathogens of various marine crustaceans.However, several studies have reported that certain strains of V. owensii and V. rotiferianus exhibit antibiotic activity against a range of human pathogens.This activity is attributed to the production of secondary metabolites, including carotenoid pigments and furan derivatives [46][47][48].Alteromonas spp.are commonly known for their role in the degradation of algal polysaccharides, which is a crucial aspect of marine carbon cycling [49].Furthermore, Alteromonas spp.have been shown to produce antifouling and antimicrobial compounds, such as hydrolyzing enzymes, questiomycins and polysaccharides [50][51][52].Lastly, isolate EPB9, identified as B. safensis, exhibited the most potent inhibitory effects against the test bacteria.
B. safensis was first identified as a contaminant from spacecraft and on the surfaces of assembly facilities [53].Since then, B. safensis has been isolated from habitats that were relatively extreme for other micro-organisms to grow in.Such habitats include rhizospheres of plants, feather dumping sites, sediments from offshore oilfields, olive oil-contaminated soil, saline desert soil and explosive-contaminated soil [54][55][56][57].Different strains of B. safensis have been shown to produce various enzymes that are important in many industrial applications, such as inulinases [57], proteases [56], keratinases [55], xylanases and pectinases [54].In addition, the presence of oxidoreductases in B. safensis and its ability to resist various metals such as Al, As(V), As (III) and Cr (VI) have been reported, which makes B. safensis a good candidate for bioremediation and biodegradation studies [58,59].Moreover, the antimicrobial activity of B. safensis has been reported in the literature.Surfactin, a biosurfactant extracted from B. safensis F4, was reported to have potent antimicrobial activity against Gram-positive bacteria such as B. subtilis and S. epidermidis.It also had moderate activity against Gram-negative bacteria such as E. coli [54].Crude extracts of B. safensis OPL 19, an endophyte of Ophioglossum reticulatum L., were also reported to have antimicrobial activity [60].The chitinase enzyme of B. safensis exhibits antifungal activity against the pathogenic Macrophomina phaseolina and Rhizoctonia solani by dissolving the cell wall component of the fungal cells [61].
Quorum sensing (QS) is a mechanism used for intra-and interspecific communication by most bacteria.This process is mediated by the production of autoinducers, which are signalling molecules that accumulate as cell density increases and, upon reaching a threshold, trigger a cascade of signal transductions that regulate many critical bacterial functions, such as sporulation, bioluminescence, virulence, reproduction and biofilm formation [62].QS is a commonly observed process in marine epibiotic relationships modulating the settlement of microbial communities on the surfaces of the host individual [63].Marine seaweeds Zone of inhibition, including the diameter of the filter paper disc (6 mm); mean value of three independent experiments±standard deviation.
a, b The different letter superscripts indicate significant difference (P<0.05) and groups that do not share the same letter are significantly different from each other.Streptomycin was used as a positive control against the test bacteria at 10 µg ml −1 .Fungisol was used as a positive control against C. albicans.
-, no activity.produce metabolites that have been shown to inhibit, stimulate and inactivate QS signals in bacteria [64][65][66].Similarly, seaweedassociated bacteria utilize QS signalling molecules to prevent the settlement of other epibionts and influence the settlement of algal zoospores [36].Accordingly, marine epiphytic bacteria present a potentially rich source of novel bioactive compounds that can be explored for drug discovery and development.In this study, C. violaceum NCTC 9757 was used as a reporter strain to screen for the anti-quorum sensing activity of the bacterial isolates from H. durvillei [10].The expression of the purple pigment, violacein, produced by C. violaceum is regulated by QS.Thus, the inhibition of violacein production is used in the present study for the preliminary screening of QSI effects.MICs and MBCs are expressed in mg ml −1 .
Our results showed that two bacterial isolates from H. durvillei, B. safensis EPB9 and B. australimaris EPB15 exhibited QSI activities against C. violaceum.Gram-positive bacteria, such as members of Bacillales, utilize peptides as autoinducers for QS, whereas Gram-negative bacteria, such as C. violaceum, use acyl-homoserine lactones (AHLs).Differences in QS molecules signify the presence of other species, and the bacteria can act on it either by translating antimicrobial compounds or by inviting them in a biofilm.QSIs can target signal detection and signalling molecules.The QSIs produced from micro-organisms affect QS of bacterial pathogens through suppression of AHL synthesis, degradation of AHL molecules and competitive binding to receptor proteins [10].Anti-virulence drugs, often known as QSIs, target SAM analogues, enzymes and antagonists in Gram-negative bacteria to block AHL formation, inactivate AHL enzymes and prevent AHL production, whereas for Gram-positive bacteria, they target apolipoprotein B to inactivate AIPs and block membrane-associated receptors [10,11,58,59].It is reported that some Firmicutes, such as Arthrobacter, Bacillus and Oceanobacillus, produce quorum-quenching enzymes that degrade AHLs and Bacillus sp.containing AHL lactonase that breaks down AHL signalling molecules [11].There are limited studies regarding the role of B. safensis as a macroalgal symbiont, but it has been shown to be a marine sponge symbiont.Thus, this study is the first to identify B. safensis as a distinct symbiont of H. durvillei.In addition, this study was the first to report the isolation and identification of B. safensis in the Philippines.However, B. safensis has been isolated from both marine and terrestrial sources from neighbouring countries such as Thailand, Taiwan, the Republic of Korea and PR China.In these studies, B. safensis was either identified as a eukaryotic symbiont or was isolated from the environment directly [68][69][70][71].
The results of this study provide baseline information regarding the epiphytic bacterial community associated with H. durvillei and their antimicrobial and QSI activities.In particular, B. safensis EPB9, one of the bacterial isolates from H. durvillei, has shown promising antibacterial properties and QSI effects.These microbial symbionts can be further explored for their various  applications in medicine and drug development.The isolation and identification of the bioactive compounds present in these bacterial isolates will reveal the underlying mechanisms behind their bioactivity.A metagenomics approach in identifying bacterial communities will further deepen our understanding of the complex relationship between H. durvillei and its associated microbiota.The Microbiology Society is a membership charity and not-for-profit publisher.
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Comments:
The work is interesting, especially in the era of antibiotic resistance, however significant changes must be made to make it scientifically sound.Abstract There is redundancy in the introduction part within the abstract, it should be concise and focused, better to move these highlights to the introduction section.Introduction: Authors should provide references of the fundamental work that sustain their statements, e.g. the use of C. violaceum to assess the antibiofilm efficiency, potential use of marine Bacillus safensis as antibiotic, in addition to the importance and accuracy of resazurin in detecting the MIC.Line#63-64: you mentioned that global warming can lead to rapid increases in AMR bacteria and environmental genes, give a brief explanation.Methods Elaborate on the molecular detection of bacterial isolates, e.g which genes were targeted?You should use the relevant appropriate and references for antimicrobial screening, MIC, MBC, and the quorum sensing inhibitory effect.What OD600 value you determined for standardising bacterial concentration for measuring the MIC.It is most important to mention the source of the bacterial and fungal test strains used in the study.Also show the if they are MDR or not.You should define their antimicrobial sensitivity to the control antibiotics used.Results You should describe the colonial morphology of bacterial isolates.Describe the SEM micrographs of bacterial isolates, especially the aggregates and biofilm structure.Line # 243, you have to distinguish the Firmicutes (Gram-positive) form the Proteobacteria (Gram negative).I suggest grouping the isolates belonging to Firmicutes together, and the Proteobacteria together, especially in tables 1-4 to make it easy for the reader to compare.Discussion Very important to provide mechanistic insights that could explain the anti quorum sensing activities of bacterial extract.In line# 235, you mentioned that" motility indicates that they form biofilms", what is the correlation between motility and biofilm formation?General Unify the referencing style The manuscript needs proofreading and editing; e.g."of note" was repeated many times, "Gram" should always start with a capital letter,…etc.

Please rate the manuscript for methodological rigour Good
Please

Fig. 2 .
Fig. 2. Representative plates showing the (a) quorum sensing inhibitory and (b) antibiotic activities of the bacterial isolates from H. durvillei against C. violaceum and E. coli, respectively.(c) The plate format was followed in determining the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the bacterial isolates against E. coli.Resazurin was added in each well after a 24 h incubation period in which blue and pink colour indicate dead and live cells, respectively.Streptomycin was used as a positive control (PC) at 10 µg ml −1 and DMSO as a vehicle control (VC).The negative control (NC) contains bacterial cells cultured in nutrient broth, whereas the sterility control (SC) only contains the media.

4 a
Zone of inhibition, including the diameter of the filter paper disc (6 mm); mean value of three independent experiments±standard deviation.a The different letter superscripts indicate significant difference (P<0.05) and groups that do not share the same letter are significantly different from each other.*Area showing visible bacterial growth without the purple coloration.Cinnamaldehyde was used as a positive control at 10 mg ml −1 .

Table 2 .
Colony morphology and Gram stain of isolated epiphytic bacteria from H. durvillei

Table 3 .
Biochemical characterization of bacterial isolates belonging to the Firmicutes group

Table 4 .
Antimicrobial activity of the EtOAc extracts of the bacterial isolates from H. durvillei at 100 mg ml −1 against test micro-organisms

Table 5 .
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the isolated epiphytic bacteria from H. durvillei against test bacteria Further studies are still needed to understand the mechanisms behind the inhibition using bacterial crude extracts of B. safensis and B. australimaris.B. safensis isolated from soil has been reported to inhibit melanin production from Cryptococcus neoformans and Cryptococcus gattii.Melanin pigments are released by cryptococcal cells through QS activity, which contributes directly to immune-modulatory activity during human infection [29].B. australimaris has been shown to have antifungal activity against Magnaporthe orayzae [67].However, QS activity of B. australimaris has not yet been reported.

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This is a study that would be of interest to the field and community.Authors have addressed reviewers' comments adequately.to the main findings of the study * The limitations of the research could have mentioned and discussed in the discussion section This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.
focusing Comments: Review comments have been addressed.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.The reviewers have highlighted major concerns with the work presented.Please ensure that you address their comments.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Methodological rigour, reproducibility and availability of underlying data: Molecular identification by 16S rDNA to the strain level by BLASTN to NCBI reference sequences carries limitations.The underlying data is publicly available and when tested, mere nucleotides separate top matches.How can the authors be confident that EPB9 is a different species or strain different than the identities of the next top two BLAST matches?It would help to confirm the species identity by another molecular or biochemical method.Can the EPb9 isolate be compared to other isolates from the same geographical area? 2. Presentation of results: Figure1C-Please add a scale bar.Figure2A,B,C-The text within the images is very small.Can it be enlarged or can larger letters and a key be provided?3.How the style and organization of the paper communicates and represents key findings: The paper title mentions B. safensis EPB9 and not the other isolates.Therefore, one expects that B. safensis EPB9 is the focus of the study.Instead, this bacterium is one of fourteen isolates and many sections in the paper do not reflect the title focus.The introduction study aims are completely different from the paper title.Please formulate a manuscript title consistent with the findings shown in the paper.4.Literature analysis or discussion: The final statement does not relate very well to the title of the paper.Is there literature precedent for B. safensis as a macoralgae symbiont?Has B. safensis been isolated in the same geographical area as the isolate in the manuscript? 5. Any other relevant comments: Lines 17, 20-Unusual sentence structures Line 39-There are no plural subjects.The use of the word "they" is unclear.'Line184-UnusualwordingPleaserate the manuscript for methodological rigour SatisfactoryPlease rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Reviewer 2 recommendation and comments https://doi.org/10.1099/acmi.0.000563.v1.5 © 2023 hagras S.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.
rate the quality of the presentation and structure of the manuscript Very goodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesReviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000563.v1.4 © 2023 Amin D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.
Dr Adnan Amin; Gomal University Faculty of Pharmacy, PAKISTAN Date report received: 15 February 2023 Recommendation: Major Revision